Treating animals with hormone preparation



United States Patent TREATING ANIMALS WITH HORMONE PREPARATION HermannKlette, Frankfurt am Main, Germany No Drawing. Application October 30,1950 Serial No. 193,042

Claims. (c1.119--1 This invention relates to hormone preparations andmore particularly to hormone preparations having the activity of thesteroid hormones, and a method of making same.

One object of this invention is to provide a hormone preparation which,although it is applied by hypodermic injection, possesses a prolongedactivity similar to that of implanted pellets and thelike.

Another object of this invention consists in providing an injectablehormone preparation which exhibits a shock effect as well as a prolongedeffect on application to the organism, said effects being controllablyadjustable.

A further object of this invention consists in provida hormonepreparation which, on account of its compo sition, is especiallysuitable in the veterinary field and may be applied, for instance, forfattening cattle, hogs and other domestic animals, for hormonalcastration of domestic animals, especially of male animals, such asboars, steers, cockerels and the like, for initiating milk secretion inheifers, sterile cows, goats, sheep and others, for sex modification inthe chick embryo, for increasing egg production, for combattingTrichomonas infections of cattle, and for various other veterinarypurposes.

Still another object of this invention consists in providing a hormonepreparation suitable in human therapy in cases in which an initiallystrong and also a prolonged effect of the hormone upon the body isrequired.

Other objects of this invention will appear hereinafter.

it is known to use in veterinary as well as human therapy solutions ofsteroid hormones in vegetable oils or in the form of implants. But theseways of application are connected with a number of disadvantage.Solutions of the hormones in oil are very quickly absorbed. Hence, theireffect lasts only for a comparatively short time. In order to obtain amore lasting effect, the injection has to be repeated quite frequently.Implantation of pellets on the other hand requires skilled personnel andis usually done by the physician himself. Although they exert aprolonged effect, one has no way to determine the exact duration of saideffect. Quite often the implanted pellets become covered with connectivetissue by which they areso so sayencysted. This results in their losingtheir activity completely because no hormone can be dissolved andabsorbed by the body fluids. Sometimes they even are discharged from thebody under suppuration. Furthermore the initial effect of said implantedpellets is only very slight and sometimes insufficient to produce thedesired therapeutic or other result. Aqueous suspensions of hormoneshave also been suggested, but the effect of the known suspensions isalso rather uncertain.

It has been found, however, that uniform, certain and prolonged effectsare achieved when applying suspen- 2,824,546 Patented Feb. 25, 1958sions of a composition according to this invention. For this purposesuspensions of said hormones are produced in which the particle size ofeach hormone particle in said suspension is chosen in such a manner thatit will produce the maximum effect. In order to achieve a strong initialeffect particles of a comparatively small particle size are employedwhile the prolonged effect is caused by particles of a comparativelylarger particle size. For producing the initial strong or shock effect,the particle size of the hormone should be not larger than 0.01 mm. indiameter while the prolonged effect is produced by particles of morethan 0.01 mm. diameter. By suitable variations of the content ofparticles of different size suspensions are obtained which may be usedfor various purposes with maximum results. The upper limit of theparticle size is determined by the width of the needle to be employed.Instead of using suspensions of particles of small particle size, onemay also employ solutions of the hormones in suitable solvents in whichthe hormone particles of larger particle size are suspended.

Suspensions according to this invention are preferably produced with thenatural follicle hormones such as estradiol, estrone, estriol, and theirderivatives, especially their esters, such as estradiol benzoate,estradioldipropionate and the like, and with synthetic compounds havingthe acivtity of said follicle hormone, for instance, compounds of thestilbene series, such as diethyl stilbestrol,di-(p-hydroxyphenyl)-hexadiene, di (p hydroxyphenyl) hexane and theirderivatives, especially their esters and ethers, but also compounds asethinyl estradiol, doisynolic acid, equilenic acid and the like, di-(p-methoxyphenyl)-phenyl ethylenebromide and others more.Di-(p-acetoxyphenyl)-hexadiene has proved of special value because, onaccount of its specific crystal structure, it is capable of formingespecially suitable suspensions.

Although compounds having the activity of the natural follicle hormonesare especially suitable for this purpose, other hormones may also beused, such as the corpus luteum hormones progesterone andethinyltestosterone, the male-sex hormones testosterone and methyltestosterone and their derivatives, especially their esters, such astestosterone propionate and the like, the adrenocortical hormones, suchas desoxy corticosterone and its acetate, corticosterone and evencortisone and other ll-substituted compounds of this type. In general,this invention is applicable to all steroid hormones and their syntheticanalogues of which an initial shock effect and at the same time aprolonged effect on application is desired.

An especially suitable field of application of the suspensions accordingto this invention is the veterinary field. It is known that, whenfattening female animals, the highest fattening effect to be achievedwith any animal in accordance with its race and age, takes place onlyafter a longer starting period during which the animals do not gainconsiderably. This is due to the fact that the sexual functions are onlygradually reduced whereby finally so-called self-castration takes placethe reason for which is, among others, luteinisation and fattydegeneration of the ovaries. With male animals the sexual instinctdisturbs also the fattening effect and diminishes the quality of theirmeat. Therefore they usually are subjected to bloody castration in orderto achieve full fattening effect.

In all these cases the application of suspensions of cornpounds havingthe activity of the follicle hormone according to this invention hasproved to be of great advantage. But it was found that for effectingcomplete and hormonal castration in the male as well as the femaleanimal definite minimum amounts of the hormonal substance have to beapplied to the animal, part of which must be very rapidly absorbable soas to cause a shock-like effect while the remainder must be absorbedgradually, i. e. in a prolonged manner. Under these circumstanceshormonal castration takes place already after a much shorter startingperiod, namely after a period of 3 to 4 weeks at the most, while whenapplying the previously used amounts of hormones said changing periodwas much longer and was about 7 to 10 weeks.

Furthermore it has been found that with said minimum effective dosis amodification in the metabolism of the treated animals takes placecausing fattening, said fattening effect being directly proportional tothe amount of hormone applied. Gains in weight are obtained therebywhich are 50-l00% higher than with control animals kept under the sameconditions. Due to the prolonged effect of the hormone suspensionaccording to this invention, the state of castration and of fattening ismaintained with female animals for any length of time and with maleanimals for about four months. Besides the extraordinary fatteningresult, the meat quality is improved because its structure becomes lessdense and fat is interspersed therein, and it acquires a lighter color.The disadvantage of the meat of male animals, especially of that ofboars, having a disagreeable specific odor and taste so that it is unfitfor human consumption, is almost completely eliminated by the treatmentwith a suspension of follicle hormones according to this invention.Thereafter the meat can be used as food.

The following minimum effective doses sufficient to cause castration andfattening in a short period of transformation were determinediSuspensions according to this invention are obtained, for instance, bysuspending previously prepared hormone particles of the desired size andin the required amounts and proportions in an aqueous medium whichpreferably contains suitable emulsifying and/or dispersing agents and,if necessary, protective colloids and the like. Especially suitable areemulsifying and dispersing agents and protective coloids which preventor at least considerably retard agglomeration of the hormone particlesand increase in size caused by growing of the crystals. Furthermore, onshaking up the suspensions before use in order to uniformly distributethe hormone particles therein, said agents must be capable of keepingthe hormone in uniform suspension for a period of time sufiicient forinjecting the same.

It is, of course, also possible to produce suspensions of the hormone inother liquids than water. Especially advantageous have provedsuspensions of coarser horrnone particles in oil solutions of the sameor a different hormone. The dissolved hormone exerts the shock-likeeffect because it is readily absorbed while the undissolved hormoneparticles produce the prolonged effect. When using water-soluble hormonederivatives, such as the estradiol glucoside phosphate compound, it ispossible to employ aqueous solutions of the same in which, for instance,estradiol benzoate which is insoluble in water, is suspended. Such asolution-suspension is also capable of exerting a powerful initialeflect upon the body whereafter the slowly absorbed estradiol benzoateparticles are maintaining the condition created by said initial action.

In order to produce hormone particles of a larger diameter than 0.01mm., the active compound is, for instance, dissolved in suitable organicsolvents, especially in ethyl alcohol or ether, so as to obtain ahot-saturated solution. By cooling while stirring slowly, the hormone isallowed to crystallize whereby the speed of cooling down the solutionand the manner of stirring are adjusted in such a manner thatsubstantially uniform crystals of a particle size of more than 0.01 mm.are obtained. The crystallized particles are separated from the motherliquor, for instance, by filtering by suction or by centrifuging or thelike, and are carefully dried under conditions whereby conglomeration ofthe particles is avoided. Thereafter the dry crystal particles are firstfreed, for instance, by sieving, wind sifting, vibration classificationor the like from particles of 0.01 mm. diameter and less and are thenseparated, if this is required, into particles of various particle size.One may also produce particles of specific and desired size by grindingdry hormone crystals in suitable mills and by subsequently sieving, windsifting and the like of the obtained powder. In order to obtain veryfine hormone particles of uniform size it is advisable to treatsuspensions of the hormones, for instance, in an aqueous medium,preferably in the presence of emulsifying and/ or dispersing agents and/or protective colloids, in a swinging mill, a colloidal mill or the likeuntil all the hormone particles have been reduced, as can be ascertainedby microscopic investigation, to substantially the same size less than0.01 mm. in diameter. A suspension in which the hormone particlespossess to a large extent such small size, is obtained, for instance, bydissolving the hormone in a suitable solvent watermiscible solvent, forinstance, in ethanol, and adding said solution while stirring vigorouslyto an aqueous solution of a suitable emulsifying and/or dispersing agentand/or protective colloid. Thereby the hormone precipitates in a veryfinely divided form because said emulsifying agents etc. preventagglomeration of the crystals. Said suspension is then. equalized insize as stated above. In order to produce a preparation according tothis invention, hormone particles of more than 0.01 mm. diameter arethen worked into said suspension in an amount necessary for the desireduse of said preparation. It is, of course, also possible, by suitablyselecting the working conditions when precipitating the hormone bypouring its alcoholic solution into the aqueous solution of anemulsifying agent etc., to obtain a preparation containing smallerparticles as well as larger ones in the desired proportion.

Another process of producing preparations according to this inventionconsists in first preparing saturated solutions of the hormone atelevated temperatures in a suitable solvent, for instance, in avegetable oil, such as olive oil, sesame oil and others, and thencooling said solution and crystallizing the hormone which at lowertemperatures does not remain in solution, under conditions wherebycrystal particles are obtained of a size corresponding to therequirements. The suspension obtained contains then part of the hormonein solution and, if necessary, part of it in the form of finer crystalsof less than 0.01 mm. diameter for shock action while part of it ispresent in the form of coarser crystals of more than 0.01 mm. diameterfor prolonged action. One may, of course, add suitable emulsifyingand/or dispersing agent's and/or protective colloids to said solutionsin order to improve the suspending power of said suspensions and toprevent conglomeration of the hormone particles.

As emulsifying and/or dispersing agents and/or protective colloids theremay be used with great advantage water-soluble cellulose ethercompounds, such as methyl cellulose or the sodium salt of carboxymethylcelluabout 1.0 to 1.5% of the same. trated solutions are too viscous fornormal use and therelose. They are used as base for the hormoneparticles.

Especially suitable have proved solutions containing More highlyconcenfore, tend to clog the hypodermic needle. Less concentratedsolutions, on the other hand, have not the emulsifying and dispersingpower necessary for keeping the hormone particles in suspension for asufliciently long period of time. Lower and higher concentrations,however, may also find proper application in specific cases.

Other agents of this type may also be used provided that they aresubstantially non-toxic and non-irritating, compatible with the body,and resorbable by the body fluids. Among them may be mentioned certainstarch fractions, like amylopectin, the polyoxyethylene sorbitanmonolaurates, monopalmitates, monostearates, monooleates which are knownby the tradenames Tween 20, 40, 60, 80 respectively, sodium cellulosesulfate, certain types of purified carrageen, which may also be added tocellulose ether solutions to increase their suspending properties,hydroxyethyl cellulose and others more.

Another way of providing a preparation which exerts a strong initial andalso a prolonged hormone effect consists in preparing pellets, tablets,small cylinders, pills and other structures which can be implantedeasily into the body, with hormone particles of different particle sizeand/or different absorbability. Such pellets etc. are composed, forinstance, of an inner core containing particles of larger size crystals,for instance, of n crystals of more than 0.01 mm. diameter, while theirouter layer covering the core partly or completely, con sists ofparticles of a size less than 0.01 mm. diameter or of crystals which arereadily soluble in the body fluids. To obtain such two part pelletsetc., one may moisten the coarser hormone particles with a concentratedsolution of a binding agent, preferably soluble in the body fluids, andmay form therefrom a core around which then may be placed in a suitablemanner a coating consisting of finer hormone particles moistenedlikewise with a concentrated solution of a binding agent which isreadily soluble in the body fluids. The outer layer may also consist ofa water-soluble hormone with a suitable binding agent. As binding agentsthere may be used cellulose ether compounds such as methyl cellulose andothers, gum acacia, sugar syrup, blood serum, lymph, and the like. Afterdrying said two part pellets under suitable conditions an implant isobtained which on application to the body will first release veryrapidly the hormone in the outer layer due to the large surface of thesmall particles or the solubility in body fluids of the water-solublehormone, thus, causing a shock effect, while the core, on account of thelarger particle size of the hormone therein, will be absorbed moreslowly, thus, effecting a more prolonged action. In principle, thecomposition of a bipartite implant according to this invention may besuch, that, after implanting the same, it must be capable to separateinto a crystal mixture consisting of single particles of different sizeand, hence, of ditferent resorbability. Implants of this type do notpossess the disadvantages of the known implants which consist of asolid, coherent mass of the hormone in question, because shortly afterimplantation they disintegrate into a crystal powder which does not tendto become covered with connective tissue as readily as a compact mass.

One very interesting application of an aqueous suspension according tothis invention is its use for modifying the sex in the embryo of chicksand other domestic birds, whereby when employing suspensions of folliclehormone, substantially only female chickens are hatched. This process ofsex modification consists in injecting an aqueous suspension of acompound having the activity of the natural follicle hormone, into theair space of the egg at an early stage in development, preferably notlater than 6 days after starting incubation. Amounts of about 20 I. U.of estrogenic activity are already sufficient to cause modification ofthe sex of the embryo in the sense of a feminisation of the hatchedchicken. With higher amounts this effect is more pronounced, but if toohigh amounts are applied the hatched animals show considerable changesof the cloaca which return to normal the older the animals become. Thebest results were obtained with hormone amounts between about 20 I. U.and 80 I. U. per egg.

It is already known to cause sex modification in the chick embryo byinjecting oily solutions of follicle hormone into the allantois part ofthe egg a few days after incubation has started. But this method cannotbe used for practical purposes; for, it is very difficult to find therather small allanteis in the egg, while injection into the air space ofthe egg according to the present invention can be carried out even by anunskilled person. In order to introduce the hypodermic needle into theegg, a small hole is first drilled into the shell, for instance, bymeans of a tooth drill whereby, however, the skin underneath the shellis not penetrated. Thereafter, the hypodermic needle is introducedthrough said hole in the shell and the skin into the air space and thehormone is injected. Preferably, not more than 0.3 cc. of suspension areapplied in which the necessary amount of hormone is suspended. Afterinjection and removal of the needle, the hole is closed by wax, paraffinor other indifferent, substantially non-toxic and non-irritatingmaterial which does not melt at incubation temperature. The eggs arethen incubated in the customary manner without further treatment. Thehatched chickens are almost all of female sex. Of course, the process isnot only applicable to hens eggs but also to eggs of other fowl, such asgeese, turkeys, ducks, and the like.

Another economically very valuable application of hormone suspensionsaccording to this inventions consists in increasing the egg productionof fowl. There are already known a number of agents which are said toincrease the egg production of fowl. In general, they comprise highlyproteinaceous fodder to which often lime or other substances capable ofpromoting the formation of the egg shell, are added. But the resultsobtained thereby are not certain but subjected to variations; Atreatment of the laying hen with a hormone preparation according to thisinvention, however, increases the egg production considerably,especially during the off-season when the egg production decreases.Furthermore it was found that the molting period is shortenedconsiderably and that egg production starts earlier in the year. Thiseffect of the hormone upon the egg production is of great importance fortwo reasons. First, eggs in larger quan tities are available in monthswith reduced egg production, and second, since each hen is capable tolay only a certain number of eggs (about 700900 eggs) during itslife-time, it is much younger at the time its egg production stops whenit has been treated with the hormone, than an untreated hen. For, theegg laying period is reduced from 3 to 4 years to 2 to 3 years. Thismeans that the meat of such a treated younger hen which is slaughteredafter egg production stops, is more tender.

An especially suitable preparation according to this invention isobtained, for instance, by impregnating and intimately mixing preferablygrain-fodder for fowls with an aqueous suspension of a follicle hormonecompound or a compound having the activity of a follicle hormonecontaining an emulsifying and/ or dispersing agent and/ or protectivecolloid and an agent capable of increasing ad herencc of the hormoneparticles to the grain-fodder, said hormone particles being of verysmall particle size. Suitable adhesive agents are the above mentionedcellulose ether compounds and the other emulsifying and/or dispersingagents, especially those which are somewhat nygroscopic and thereforequite sticky. But other substances may be used likewise, such as gumacacia, algiru'c 7 acldgand other mucilaginous extracts and the like,pro vided they are non-toxic and non-irritating to the animal. Ofcourse, it is also possible to impregnate the fodder with a solution ofthe hormone in a suitabl solvent, thereby adding preferably an agentcapable of increasing adherence of the hormone particles to the fodder,whereafter the impregnated fodder is intimately mixed, if necessary,with further amounts of fodder so as to guarantee uniform distributionof the hormone throughout the fodder.

One may also produce aqueous solutions of watersoluble hormonepreparations and add the same in the required amounts to the drinkingwater. furthermore, one may include the hormone into fodder as it isused for cramming poultry. This manner of application has the advantagethat each bird receives a predetermined amount of hormone. One may alsoapply hormone suspensions according to this invention by injection,whereby the advantage is attained that considerably smaller amounts ofthe hormone are required. Any other suitable way of applying the hormonemay also be employed.

The amounts of hormone to be given daily depend, of course, upon thekind of fowl and the various races to be treated. Furthermore, theresorbability and the solubility of the hormone used play also animportant role. The diacetate of di-(p-hydroxyphenyl)-hexadien has beenproved of special advantage. Of this compound an amount of about4000-6000 I. U. daily has given very satisfactory results. Of course,other hormone compounds may be used likewise.

Suitable local anesthetics as well as preserving agents may be added tothe suspensions and other preparations provided these additions do notirritate the body and do not react or in any way affect the propertiesof the active hormone ingredient. As preserving agents there may beused, for instance, chlorobutanol, phenol, cresol, thimerosal[sodium-(ethylmercuri)-thiosalicylate], n-butyl-phy'droxybenzoate andothers while benzocaine, butacaine sulfate, tetracaine hydrochloride andthe like may be used as local anesthetics.

Instead of using water as base for preparing the hormone suspensionsaccording to this invention, isotonic sodium chloride solution, Ringerssolution, blood serum and others may be used likewise.

The term hormone as used throughout the specification and the claimsannexed hereto indicates not only the actual natural hormones but alsosynthetic chemical compounds and their derivatives having the activityof said natural hormones.

The following examples serve to illustrate the invention without,however, limiting the same to them.

Example 1 0.5 g. of amylopectin are dissolved in 100 cc. of water,10,000,000 I. U. of estradiolglucosido phosphate are added thereto andare dissolved therein by heating while stirring. After cooling thesolution to room temperature, 10,000,000 I. U. of the dimethyl ether ofdi-(p-hydroxyphenyl)-hexadiene crystals having a particle size ofbetween about 0.05 and 1.00 mm. diameter are added slowly while stirringvigorously to said solution. A suspension of hormones is obtained whichcontains part of the hormones in dissolved and, thus, readily andrapidly absorbable form while the remainder of the hormones is presentin coarsely crystalline form which is absorbed only gradually and veryslowly.

Hormone crystals of between about 0.05 and 1.0 mm. diameter are obtainedby dissolving the above mentioned methyl ether in methanol so that ahot-saturated solution is formed, and allowing the solution to coolwhile stirring slowly whereby part of the ether crystallizes. Thecrystals are then filtered, dried, carefully ground, and separated bysieving into crystals of about 0.05 to 1.0 mm. diameter.

Example 2 10,000,000 1. U. of di-(p-hydroxyphenyl)-hexadien aredissolved in cc. of olive oil (U. 5. Ph. )GV) while heating to about C.and stirring continuously. The solution is then cooled within 5 hours toabout 0 C. while stirring slowly, is kept at this temperature for about3-4 hours and is then slowly allowed to attain room Example 3 4 g. ofdiethyl stilbestrol (40,000,000 I. U.) are dissolved in 10 cc. ofboiling ethanol and are gradually added to 40 cc. of a solution of 1% ofmethyl cellulose in water whereby the mcthylcellulose solution isstirred by means of a very effective stirring apparatus. The hormone isthereby precipitated in finely divided form. The suspension obtained ishomogenized in a swinging mill until most of the particles show underthe microscope a particle size of about 0.001 mm. to about 0.005 mm.diameter.

6 g. of diethyl stilbestrol crystals having a particle size of betweenabout 0.05 mm. to about 0.15 mm. diameter, as they are obtained byallowing a hot-saturated alcoholic solution of said hormone tocrystallize, filtering off the crystals by suction, drying them, andseparating crystals of said size by sieving, are slowly and graduallyadded to 60 cc. of a 1.5% solution of a mixture of equal parts of methylcellulose, known by the tradename Tylose S 400, and of the sodium saltof carboxymethyl cellulose, known by the tradename Tylose K 2000, inwater while stirring thoroughly until a uniform suspension of thehormone is obtained.

Both suspensions are mixed with each other while stirring thoroughly. Asuspension is obtained containing about 40% of the hormone having aparticle size of about 0001-0005 mm. diameter and about 60% of thehormone having a particle size of about 0.05-0.15 mm. diameter.

By mixing such previously prepared hormone suspension in any desiredproportion, suspensions of various composition are obtained which may beemployed for many purposes. They are readily applicable by hypodermicinjection and allow the application of very small as well as very largehormone doses of a composition adapted to any desired use.

With suspensions obtained according to the above given examples or in asimilar manner the following results were obtained.

A hormone suspension containing at least 50% of very finely dividedparticles is subcutaneously injected behind the ear. Experiments werecarried out, for instance, with non-pregnant sows, 7 months to 2 yearsold. The animals did not grow properly and did not respond well tofattening fodder. They were frequently and strongly in heat. Within l-2days after the injection ruttish animals calmed down and did not exhibitany heat during the following fattening period while with animals whichwere free of heat at the time of injection, ruttishness appeared which,however, disappeared after 4-5 days. When applying at least 60,000 I. U.hormone per kg. live weight, about l8-21 days were required to effectthe necessary change in the endocrine system. Thereafter the fatteningeffect proper sets in and the animals gain considerably more in weightthan untreated animals under like conditions. The average increase ingain amounted to 50-.100% more than with the controls. Sows of 70 kg.weight, for instance, were injected with about 5,000,000 I. U. of asuspension of the diacetate of di-.(p-hydroxyphenyl)-hexadiene, whilesows with about kg. live weight received about 10,000,000 I. U. of thesame suspension and sows with about- 200 kg. live weight about15,000,000 1. U. The

following table shows the fattening effect of these animals incomparison with untreated controls:

This table shows furthermore that, in order to obtain about the samefinal weight, the fattening duration can be reduced 30-50%. A furthergreat advantage of the treatment is as stated above, the considerableimprovement of the meat quality due to the interspersion of fat in themuscle. The meat-fat-ratio, however, remains in general normal. Bloodycastrated boars respond to the treatment in about the same manner assows, while with not castrated bears the results were less uniform. Ahigher increase in weight of -100% over that of untreated boars wasachieved by the injection of a hormone suspension according to thisinvention. With these animals the great advantage is achieved that, asstated above, their meat becomes fit for human consumption. It loses itspeculiar odor and taste about 4-6 weeks after the injection. Thecastration effect of the hormone, however, lasts only for about 3-4months. Hence, the treatment should be carried out about 3 months beforethe animal will be slaughtered.

With wethers, especially young animals, a gain of about 30% more thanwith untreated animals is observed, but only half the amount of hormoneper kg. live weight is required than with sows and boars.

With oxen and cows an additional gain of about 30-70% over untreatedanimals is observed. Compare, for instance, the following table, wherebythe animals of 200 kg. of weight received about 5,000,0007,000,000 I. U.of the hormone while those of 400 kg. of weight were injected with about10,000,00014,000,000 I. U. of the hormone:

With cows similar results were obtained. Other experiments showed thefollowing results:

Average daily increase in weight with animals treated with a hormonesuspension according to this invention:

Treated. Controls, More inanimals, g. crease,

percent Oxen 897 660 36. O Steers 1, 141 710 60. 8 Cows and Heifo 983750 31. 1

Hence, the treated animals could be slaughtered about one month earlierthan the controls.

Another field of application of hormone suspensions is the hormonalcaponizing of fowls. This process has considerable advantages over thebloody castration. First of all, no losses due to the castrationoperation occur. And the speed of hormonal castration is much higherthan that by surgical means. The following table gives the comparativeresults of bloody castrated animals and of hormonal castrated ones:

Weight at Weight after beginning of experiment, g. 2 weeks, 4 weeks, 6weeks,

g. g. g.

Bloody castration 795 1, 032 1, 246 1, 418 Hormonal oastration 795 8661, 416 1, 754

This table shows that, although in the beginning the gain in weight isnot as high with hormonal castration than with bloody castration, thepicture has entirely changed after 6 weeks, when the hormonal caponsshow a 23.7% higher weight than the bloody castrated ones. The overallincrease in weight is with the former about with the latter about 78%.

A further possibility of utilizing the hormone suspensions exists forpromoting milksecretion in heifers, sterile cows, goats, and sheep. Withgoats positive results could be achieved in almost all cases. Sterilegoats gave by one injection with 2,000,0002,500,000 I. U. hormone afterabout 14 days to 3 weeks about 1.5-2.5 liters of milk daily. The milkproduction remained almost the same for about 7-8 months. With cows ingeneral the results are not as certain; but in some cases the'milkproduction amounted to 12 liters daily. Amounts of 17 liters daily werealso reported.

Hormone suspensions containing a large percentage of very smallparticles of a size of 0.001-001 mm. diameter may be successfully usedinstead of oil solutions of the hormone, for instance, for the treatmentof trichomonas infection of cattle and in other cases where a verystrong initial effect and a comparatively short prolonged eifect isdesired.

Example 4 A mixture of roughly ground corn germs and wheat bran 1:1 isspread in a thin layer upon a plate and is then sprayed with asuspension of di-(acetoxyphenyl)- hexadien in an aqueous 0.5% methylcellulose solution so that 5 g. of the impregnated material containsabout 5,000 I. U. of the hormone. The sprayed mass is then furthermoreground and intimately mixed so as to obtain a preparation throughoutwhich the hormone is uniformly and evenly distributed. 5 g. of thepreparation added daily, preferably in the morning, to the fodder ofeach laying hen increases the egg production of said hen in theoff-season to about 50%.

Example 5 19.0 g. of finely ground oats, 15.0 g. of wheat middlings,10.0 g. of wheat bran, and 5.0 g. of soybean meal are thoroughly mixedwith each other. This mixture is sprayed with a concentrated alcoholicsolution of 100,000 I. U. of di-(p-hydroxyphenyl)-hexadien in a mixerwhich can be heated to 50 C. while an air stream is blown through it.The alcohol is evaporated thereby and recovered in the customary manner.The impregnated mass is then intimately mixed with 10.0 g. of dried skimmilk, 10.0'g. of meat scrap containing 55% of protein, 15.0 g. ofalfalfa-leaf meal, 4.0 g. of linseed meal, 5.6g. of ground limestone,2.4 g. of streamed bonemeal, 1.2 g. of a salt mixture containing 100parts of common salt and 1.7 parts of anhydrous manganous sulfate, and2.8 g. of cod-liver oil, until the hormone is uniformly and evenlydistributed therein. 5 g. of this laying mash is fed daily to eachlaying hen.

Example 6 A paste-like mass containing only very little water is made bymoistening estradiol benzoate crystals having a particle size of about0.1-0.3 mm; diameter, with a concentrated aqueous solution of gum acaciawhereby the amount of binding agent is just high enough to cause thesingle hormone crystals to stick to each other without filling out allthe interstices betweensaid crystals. When preparing this paste care hasto be taken that the particles are not substantially broken up intosmaller particles.

Likewise another paste-like mass is produced by binding estradiolcrystals having a particle size of about 0.001-0.005 mm. diameter with aconcentrated aqueous solution of methyl cellulose.

The pastes are then brought into extrusion machines the head of which isprovided with two orifices, one in the midle forming a core and theother surrounding concentrically the former, thus, forming a tube which,on

' account of the adhesiveness of the paste sticks to the inner core andforms a cover around it. The mass containing the coarse estradiolbenzoate crystals is forced through the inner orifice while the masscontaining the fine estradiol crystals is forced through the outer con-Example 7 A tablet of 1.5 mm. diameter and 1.5 mm. height is pressedfrom di-(p-aeetoxyphenyl)-hexadien crystals having a particle size ofabout 0.2-0.3 mm. diameter and a suitable binding agent, such as carboxymethyl cellulose sodium, whereby care is taken that the pressure is nottoo high but that the crystals substantially maintain their size andshape and are not reduced in size to powder. This tablet is then used asa core around which in a manner known per se a coating is appliedconsisting of a concentrated sugar syrup containing di-(p-acetoxyphenyl)-hexadien crystals having a particle size of about0.001-0.005 mm. diameter. Thereby pellets are obtained consisting of acore of coarse crystals and a coating of fine crystals which can beimplanted quite easily.

Of course, many changes and variations may be made by those skilled inthe art in the reaction conditions, the

hormones, binding, emulsifying, dispersing, adhesive agents, solventsemployed, the particle sizes of the hormone, the preparation of hormonecrystals of specific particle size, and the like, provided these changesand variations are made in accordance with the principles set forthherein and the claims annexed hereto.

What I claim is:

1. In a method of increasing body growth and improving meat quality ofcattle and other domestic animals, the step comprising administering tosaid animals, by a single non-intravenous injection, a suspension of anestrogen compound in an injectable vehicle, said suspension containingestrogen particles of a size substantially between about 0.001 mm. andabout 0.005 mm. diameter and estrogen particles of a size substantiallybetween about 0.05 mm. diameter and about 0.50 mm. diameter.

2. In a method according to claim 1, wherein the finer estrogenparticles and the coarser estrogen particles are present in thesuspension in about equal amounts by weight.

12 3. In a dosage procedure for increasing body growth and improvingmeat quality of cattle and other domestic animals, the steps comprisingadministering to said animals, by a single non-intravenous injection, :1suspension of an estrogen compound in an injectable vehicle,

said suspension containing estrogen particles of a size between about0.001 mm. and about 0.005 mm. diameter and estrogen particles of a sizebetween about 0.05 mm. and about 0.50 mm. diameter, the finer and thecoarser particles being present in said suspension in about equalamounts, the total amount of estrogenic compound being injectedcorresponding to at least the following estrogenic international unitsper kg. of live weight of said animal:

I. U. per kg. ltvc Bulls Cows, steers, heifers" Boa ams 20,000 Sheep15,000. Goats, male 25,000. Goats, female. 18,000. Old cocks, hen600,000 per animal. Coekerels.- 350,000 per animal.

700,000 per animal.

and keeping said treated animals on ordinary feed until slaughteringtime.

4. In a method according to claim 3, wherein the estrogenic compound isdi-(p-acetoxy phenyl) hexadiene.

5. In a method according to claim 1, wherein the estrogen compound isdi-(p-acetoxy phenyl) hexadiene.

References Cited in the file of this patent UNITED STATES PATENTS1,928,346 Axelrod Sept. 26, 1933 2,142,537 Tisza Jan. 3, 1939 2,207,990Miller July 16, 1940 2,343,625 Abramson et al. Mar. 7, 1944 2,385,117Turner et a1 Sept. 18, 1945 2,394,628 Meyer Feb. 12, 1946 2,413,419Saunders Dec. 31, 1946 2,486,426 McGaha Nov. 1, 1949 2,541,447 TurnerFeb. 13, 1951 2,557,052 Himelick June 12, 1951 2,589,898 Turner Mar. 18,1952 FOREIGN PATENTS 417,715 Great Britain Oct. 1, 1934 515,566 GreatBritain Dec. 8, 1939 543,897 Great Britain September 1941 OTHERREFERENCES Forbes: Article in Science, Apr. 21, 1941, pp. 404-405.

Greene: Abstracts of Surg. Gym. and Obst, June 1942, pp. 595-599, p. 596is especially pertinent.

J. of Endocrinology, vol. 4, 1944-1945, pp. 51 and 77.

Koref et al.: Article in Endocrinology, Mar. 1946, pp. 214 and 215.

Bowling et al.: Article in J. A. M. A., Nov. 1, 1947, pp. 567-569.

Squibb Abst. Bulletin, July 19, 1950.

1. IN A METHOD OF INCREASING BODY GROWTH AND IMPROVING MEAT QUALITY OFCATTLE AND OTHER DOMESTIC ANIMALS, THE STEP COMPRISING ADMINISTERING TOSAID ANIMALS, BY A SINGLE NON-INTRAVENOUS INJECTION, A SUSPENSION OF ANESTROGEN COMPOUND IN AN INJECTABLE VEHICLE, SAID SUSPENSION CONTAININGESTROGEN PARTICLES OF A SIZE SUBSTANTIALLY BETWEEN ABOUT 0.001 MM. ANDABOUT 0.005 MM. DIAMETER AND ESTROGEN PARTICLES OF A SIZE SUBSTANTIALLYBETWEEN ABOUT 0.05 MM. DIAMETER AND ABOUT 0.50 MM. DIAMETER